SUMMARY Hookworm infection remains one of the most important public health threats worldwide, with an estimated 800 million people infected. Heavy hookworm infection is the leading cause of anemia in the tropics, resulting in debilitating and sometimes fatal iron-deficiency anemia caused by blood loss to feeding adult worms in the intestine. Children, pregnant women, and the elderly are particularly susceptible to morbidity from hookworm infection. Control strategies are restricted to periodic de-worming of infected individuals with benzimidazole (BZ) anthelmintics. There is considerable concern that resistance to BZ drugs will develop with increased use in mass drug administration (MDA) programs to control hookworms. Resistance is also a major concern for other drugs such as ivermectin (IVM) and pyrantel (PYR) which are commonly used to treat lymphatic filariasis and soil transmitted nematodes. Molecular tests to monitor the emergence of resistance are necessary, but the genetic mutations that confer resistance to BZ, PYR and IVM are unknown in hookworm. Molecular tests can help tailor drug treatments during MDA to be more effective, but first we need to identify mutations that can emerge in the wild and lead to resistance. We have identified a strain of hookworm Ancylostoma caninum that exhibits resistance to BZ, PYR, and IVM. This is the first known strain of hookworm that is resistant to three drugs, as well as the first resistant strain resistant to both TBZ and IVM. This strain was not developed in a laboratory and so represents a unique opportunity to understand the genetics of naturally emerging resistance. We propose to use this strain to identify the underlying mechanisms of resistance, and to determine if resistance to one drug selects for resistance to the others. In Aim 1, we propose to conduct massively parallel sequencing of resistant recombinants from a cross between our resistant strain (termed KGR) and the wildtype (susceptible) strain (termed WMD). We will search for areas of high SNP density from the resistance parental strain within the F2 recombinant strain, so as to identify the causative genetic lesions. In Aim 2, we will use Caenorhabditis elegans as a surrogate to test the sufficiency of identified mutations from Aim 1 in conferring resistance. This proposed research plan would represent the first ever example of recombinant SNP mapping in hookworm, and will reveal novel, naturally emerging markers of AR. These markers will permit the development of molecular based tools to monitor the frequency of resistance alleles in populations undergoing treatment in a timeframe that permits the implementation of steps to prevent widespread resistance from developing in treated hookworm populations.